The most common methods for in Vitro cultivation of adult hepatocytes do not allow long-term survival neither expression of their differentiated functions. In well known methods, hepatocytes can be cultivated in tissue culture dishes for only 3-8 days after which they do not show characteristic morphology neither express their differentiated functions. (For references see G. Michalopoulos and H. C. Pitot, Exptl. Cell Res., 94, 70, 1975).
Attempts have been made to extend the survival of the cultivated hepatocytes by improving both the culture medium (G. M. Williams, E. Bermudez, R. H. C. San, P. J. Goldblatt, and M. F. Laspia, In Vitro, 14, 824, 1978; P. E. Schwarse, A. E. Solheim, and P. O. Seglen, In Vitro, 18, 43, 1982) and the substratum of the culture dishes to which liver cells have to attach. Hepatocytes have been cultivated on collagen coated dishes and collagen membranes (G. Michalopoulos, and H. C. Pitot, Exptl. Cell Res., 94, 70, 1975), nitrocellulose filters (C. R. Savage, and R. J. Bonney, Exptl. Cell Res., 114, 307, 1978), collagen gel-coated nylon meshes (A. E. Sirica, W. Richards, Y. T. Sukada, C. A. Sattler, and H. C. Pitot, Proc. Natl. Acad. Sci, USA, 76, 283, 1979), fibronectin coated dishes (J. Deschenes, J. P. Valet, and N. Marceau, In Vitro, 16, 722, 1980), dishes with extracellular matrix produced by bovine corneal endothelial cells (C. Guguen-Guillouzo, D. Seignoux, Y. Courtois, P. Brissot, N. Marceau, D. Glaise, and A. Guillouzo, Biol, Cell., 46, 11, 1982), or with connective tissue biomatrix obtained from liver (M. Rojkind, Z. Gatmaitan, S. Mackensen, M. A. Giambrone, P. Ponce, and L. Reid, J. Cell Biol., 87, 225, 1980). Although epithelial morphology and some differentiated functions are conserved for longer periods, changes in genetic expression occur in some cases leading to foetal-like phenotype.
Another useful approach for cultivating epithelial cells is their coculture with feeder cells; these feeder cells are x-irradiated being unable themseleves to multiply, but they exhibit active metabolism (T. T. Puck, P. I. Marcus and S. J. Cieciura, J. Exp. Med., 103, 273, 1956). Incubation of feeder cells with mitomycin-C gives the same results (I. MacPherson and A. Bryden, Exp. cell Res. 69, 240, 1971; J. Taylor Papadimitriou, M. Shearer and M. G. P. Stocker, Int. J. Cancer, 20, 903, 1977; J. G. Rheinwald, Meth. Cell Biol., 21, 229, 1980). Epithelial cells have been therefore cocultured with feeder cells, like 3T3 murine fibroblasts (J. G. Rheinwald and H. Green Cell, 6, 331, 1975) or other types of murine 3T6 or human fibroblasts (J. Taylor-Papadimitriou, M. Shearer, and M. G. P. Stocker, Int. J. Cancer, 20, 903, 1977; R. C. Armstrong and W. Rosenau, Cancer Res., 38, 894, 1978; A. L. Epstein, and H. S., Kaplan, Cancer Res. 39, 1748, 1979).
However, hepatocyte coculture with human lung fibroblasts (G. Michalopoulos, F. Russell, and C. Biles, In Vitro, 15, 796, 1979) or with mouse embryo C3H/10T1/2 cells (R. Langenbach. L. Malick, A. Tompa, C. Kuszynski, H. Freed, and E. Huberman, Cancer Res., 39, 3509, 1979) does not result in a significant long-term survival or maintenance of hepatic characteristics. Although hepatocytes cocultured with viable liver epithelial cells (C. Guguen-Guillouzo, B. Clement, G. Baffet, C. Beaumont, E. Morel Chany, D. Glaise, and A. Guillouzo, Exp. Cell Res., 143, 47, 1983) survive for long periods and produce albumin, their capacity for chemical hepatotoxicity testing has not been determined.
Since interaction of hepatocyte precursor cells with mesenchyme or mesenchyme products seems essential for liver development in chicken (N. Le Douarin, Developmental Biology, 17, 101, 1968), we used 3T3 fibroblasts, mesenchyme derived cells, to support the long-term survival and the expression of differentiated functions of adult rat hepatocytes in culture.